Repurposing picropodophyllin as a potential thyroid eye disease treatment via delaying mitotic clonal expansion through a patient‐derived preclinical platform

Dear Editor, Thyroid eye disease (TED), the most frequent orbital disease and the leading cause of proptosis in adults, is a debilitating, disfiguring, and even sight-threatening disease.1 Drugs as alternative options are required for TED patients who need surgery to control orbital compression. Targeting the enhanced adipogenesis of orbital tissue removed by surgery has therapeutic potential for TED by inhibiting the adipogenesis of orbital fibroblasts (OFs). We have developed lenvatinib as a novel therapeutic option for TED based on the primary OFs in-vitro model.2 However, translational research is hampered by the limited number of OFs and the lack of reproducible preclinical platforms for drug discovery.3 Thus, we generated a TED patient-derived orbital fibroblast cell line (OF-CL) with proliferative and adipogenic capacity, which was further applied in high-throughput drug screening systems, 2D and 3D in vitro models, and xenograft in vivo models for drug repurposing in TED without a dramatic loss of inherited genotypes. Taking advantage of drug repurposing, we identified picropodophyllin (PPP), recognized as an inhibitor of insulin-like growth factor-1 receptor (IGF-1R) and in clinical phase II in lung cancer, with anti-adipogenic therapeutic potential for TED. The OF-CL was established through immortalisation of OFs that were isolated from the orbital adipose tissue of TED patients (Figures S1 and 1A). It possessed a fibroblast-like morphology and the enhanced proliferative capacity (Figure 1B,C). RNA-sequencing analysis indicated proliferation characteristics at the transcriptomic level (Figures 1D,E and S2A,B). We also evaluated the adipogenesis capacity of OF-CL and OFs (Figure 1F). Brightfield images and Bodipy staining showed similar formations of intracellular lipid droplets during adipogenic differentiation (Figures 1G,H and S2C), which were confirmed by RNA-sequencing analysis and qPCR test with genes involved in the formation of mature

Dear Editor, Thyroid eye disease (TED), the most frequent orbital disease and the leading cause of proptosis in adults, is a debilitating, disfiguring, and even sight-threatening disease. 1 Drugs as alternative options are required for TED patients who need surgery to control orbital compression. Targeting the enhanced adipogenesis of orbital tissue removed by surgery has therapeutic potential for TED by inhibiting the adipogenesis of orbital fibroblasts (OFs). We have developed lenvatinib as a novel therapeutic option for TED based on the primary OFs in-vitro model. 2 However, translational research is hampered by the limited number of OFs and the lack of reproducible preclinical platforms for drug discovery. 3 Thus, we generated a TED patient-derived orbital fibroblast cell line (OF-CL) with proliferative and adipogenic capacity, which was further applied in high-throughput drug screening systems, 2D and 3D in vitro models, and xenograft in vivo models for drug repurposing in TED without a dramatic loss of inherited genotypes. Taking advantage of drug repurposing, we identified picropodophyllin (PPP), recognized as an inhibitor of insulin-like growth factor-1 receptor (IGF-1R) and in clinical phase II in lung cancer, with anti-adipogenic therapeutic potential for TED.
The OF-CL was established through immortalisation of OFs that were isolated from the orbital adipose tissue of TED patients ( Figures S1 and 1A). It possessed a fibroblast-like morphology and the enhanced proliferative capacity ( Figure 1B,C). RNA-sequencing analysis indicated proliferation characteristics at the transcriptomic level ( Figures 1D,E and S2A,B). We also evaluated the adipogenesis capacity of OF-CL and OFs ( Figure 1F). Brightfield images and Bodipy staining showed similar formations of intracellular lipid droplets during adipogenic differentiation ( Figures 1G,H and S2C), which were confirmed by RNA-sequencing analysis and qPCR test with genes involved in the formation of mature adipocytes and lipid synthesis including (C/EBP)α, fatty acid-binding protein 4 (FABP4), perilipin 1 (PLIN1), peroxisome proliferator-activated receptor gamma (PPARγ) and adiponectin (ADIPOQ) ( Figure S2D-F). 2 Drug repurposing is a safe and successful strategy to speed up the drug discovery and development processes for existing drugs. 4 To identify candidates for drug repurposing in TED adipogenesis upon patient-derived OF-CL, we first hand-curated a collection of 153 small molecule compounds targeting genes involved in adipogenesis pathways of white adipose tissue from registered clinical trials (Table S3). 5 To speed up the screening, we set up a quantitative high-throughput screening system based on patient-derived OF-CL to test the rate of anti-adipogenesis of these 153 compounds via Bodipy staining ( Figure 2A). As shown in Figure 2B, although there were no beneficial effects on the anti-adipogenesis of TED by several compounds that have been documented to inhibit adipogenesis in mesenteric adipose tissue, such as resveratrol and genistein. We identified 25 candidates with antiadipogenic therapeutic potential for TED (Table S1). The main documented targets of these compounds was defined as vascular endothelial growth factor receptor (VEGFR), c-Kit, IGF-1R, mammalian Target of Rapamycin (mTOR), p38 mitogen-activated protein kinase (p38 MAPK), COX, etc.
PPP among 25 candidates is in first and second phase clinical trials of lung cancer 6 and shares the IGF-1R target with teprotumumab antibody, which is the first and only Food and Drug Administration-approved drug of TED. 7 To validate the in vitro inhibitory effect of PPP on adipogenesis of TED, we investigated the anti-adipogenesis effect of PPP on primary OFs. It showed a dose-dependent decrease in intracellular lipid accumulation and adipogenesisrelated mRNA expression ( Figure 2C-E).
To explore the anti-adipogenesis mechanisms of PPP, we investigated the adipogenesis stages disturbed by PPP

F I G U R E 1 Generation and assessment of a human orbital fibroblast cell line (OF-CL) from a patient with thyroid eye disease (TED). (A)
Schematic view of the construction of an orbital adipose tissue (OAT)-derived OF-CL from a TED patient. The stromal vascular fraction (SVF) was isolated from the OAT of a 35-year-old female TED patient who underwent decompression surgery, and cultured in dishes for primary orbital fibroblasts (OFs). SV40 large T antigen was transformed into OFs using a retroviral system to generate the immortalised OF-CLs. (B) Brightfield microscopy photo of OFs and OF-CL (bar = 200 μm). (C) Doubling time of different generations of OFs and OF-CL (*p < .05, **p < .01). (D) Volcano plot showed differentially expressed genes (DEGs) between OFs and OF-CL with absolute fold change ≥2 and false discovery rate <0.05 (n = 3 for each cell type). Blue and red dots indicate upregulated DEGs in the OF and OF-CL groups, respectively. All other genes are labelled as grey dots. (E) Gene Ontology (GO) analysis of DEGs showed that proliferation-related signalling pathways were enriched in OF-CL. (F) Schematic timeline of adipogenic differentiation and assessment in the 2D culture of OFs or OF-CL. A total of 8 × 10 3 cells were plated into each well of 96-well plates, and grew to 100% confluence in growth medium on day −3. Cells were cultured for another 3 days in normal growth conditions before induction medium was employed on day 0, and the medium was refreshed every 3 days. The medium was changed into the maintenance medium on day 6 and refreshed every three days. RNA extraction was conducted on the day 6 for the detection of adipogenic differentiation marker genes. An assessment of lipid accumulation was performed on day 14. (G) Bodipy and 4',6-diamidino-2-phenylindole (DAPI) staining indicated the intracellular lipid droplets of adipocytes on day 14 (bar = 50 μm). (H) Quantitative analysis of relative Bodipy intensity (***p < .001).   ( Figure 3A). 8 PPP suppressed expression of C/EBPδ and C/EBPβ in the early stage, C/EBPα and PPARγ in the intermediate stage and FABP4 and ADIPOQ in the late stage ( Figures 3B and S4). During early stage of differentiation, preadipocytes were reported to undergo mitotic clonal expansion (MCE) with about two rounds of division to proliferation. 9 C/EBPδ and C/EBPβ are the core transcription factors of MCE. To investigate whether PPP affects early proliferation, we observed a reduction in cell number in the PPP-treated group at 48 and 96 h ( Figure 3C) and a decrease in proliferation indicated by EdU-positive cells ( Figure 3D,E). The MCE regulatory genes, including cyclin-dependent kinase (CDK)4, CDK6 and Cyclin D1, in the first 24 h of adipogenesis were also suppressed by PPP at transcriptional level ( Figure S5A).
To further determine the specific phases of the cell cycle blocked by PPP during MCE, ploidy analysis was performed to analyse the cell distribution at 24 and 16 h after differentiation initiation. Phosphorylate histone H3, M phase cell marker, was used to distinguish cells in G2 phase or M phase (Figures 3F-I and S5B-F). These data suggest that PPP may cause G2/M arrest and inhibit the MCE process ( Figure 3J).
Three-dimensional culture models provide an alternative to 2D cell culture to closely mimic the cellular microenvironment in diseases. 10 To assess the antiadipogenesis function in 3D culture systems, we employed sodium carboxymethyl cellulose and Matrigel to form a fibroblast spheroid ( Figure 4A,B). After 21-day adipogenic differentiation, PPP suppressed 68.31% of lipid accumulation in spheroids of OFs compared with control ( Figure 4C,D). The mRNA levels of adipogenic marker genes were significantly reduced in the PPP group ( Figure S6A).
To address the challenge of TED rodent models for drug validation, we carried out a cell line-based xenograft model in humanised immunodeficient mice by subcutaneously injecting OF-CL ( Figure 4E). PPP reduced 50% of the adipose tissue volumes generated from the OF-CL implantations after the 21-day growth period (Figure S5F,G). Immunofluorescence and haematoxylin and eosin staining showed that the PPP group had fewer adipocytes observed and reduced fat vacuole formation in the implantation sites compared with control group (Figures 4H, 4I and S6B,C).
Our study developed an easy-to-use and reproducible platform with high-throughput drug screening, a 3D culture model and an in-vivo model for TED studies, which was based on a TED patient-derived OF-CL with proliferative and adipogenic properties and without severely perturbing genotype inheritance. Through the platform, we found that PPP could suppress adipogenesis by restraining MCE. Future studies can take advantage of this patient-derived platform to discover drugs for TED and explore PPP outcomes in patients.